RESUMO
The primary function of dystrophin is to form a link between the cytoskeleton and the extracellular matrix. In addition to this crucial structural function, dystrophin also plays an essential role in clustering and organizing several signaling proteins, including ion channels. Proteomic analysis of the whole rodent brain has stressed the role of some components of the dystrophin-associated glycoprotein complex (DGC) as potential interacting proteins of the voltage-gated Ca2+ channels of the CaV2 subfamily. The interaction of CaV2 with signaling and scaffolding proteins, such as the DGC components, may influence their function, stability, and location in neurons. This work aims to study the interaction between dystrophin and CaV2.1. Our immunoprecipitation data showed the presence of a complex formed by CaV2.1, CaVα2δ-1, CaVß4e, Dp140, and α1-syntrophin in the brain. Furthermore, proximity ligation assays (PLA) showed that CaV2.1 and CaVα2δ-1 interact with dystrophin in the hippocampus and cerebellum. Notably, Dp140 and α1-syntrophin increase CaV2.1 protein stability, half-life, permanence in the plasma membrane, and current density through recombinant CaV2.1 channels. Therefore, we have identified the Dp140 and α1-syntrophin as novel interaction partners of CaV2.1 channels in the mammalian brain. Consistent with previous findings, our work provides evidence of the role of DGC in anchoring and clustering CaV channels in a macromolecular complex.
Assuntos
Distrofina , Proteômica , Animais , Distrofina/genética , Distrofina/metabolismo , Mamíferos/metabolismo , Neurônios/metabolismoRESUMO
The mdx52 mouse model of Duchenne muscular dystrophy (DMD) is lacking exon 52 of the DMD gene that is located in a hotspot mutation region causing cognitive deficits and retinal anomalies in DMD patients. This deletion leads to the loss of the dystrophin proteins, Dp427, Dp260 and Dp140, while Dp71 is preserved. The flash electroretinogram (ERG) in mdx52 mice was previously characterized by delayed dark-adapted b-waves. A detailed description of functional ERG changes and visual performances in mdx52 mice is, however, lacking. Here an extensive full-field ERG repertoire was applied in mdx52 mice and WT littermates to analyze retinal physiology in scotopic, mesopic and photopic conditions in response to flash, sawtooth and/or sinusoidal stimuli. Behavioral contrast sensitivity was assessed using quantitative optomotor response (OMR) to sinusoidally modulated luminance gratings at 100% or 50% contrast. The mdx52 mice exhibited reduced amplitudes and delayed implicit times in dark-adapted ERG flash responses, particularly in their b-wave and oscillatory potentials, and diminished amplitudes of light-adapted flash ERGs. ERG responses to sawtooth stimuli were also diminished and delayed for both mesopic and photopic conditions in mdx52 mice and the first harmonic amplitudes to photopic sine-wave stimuli were smaller at all temporal frequencies. OMR indices were comparable between genotypes at 100% contrast but significantly reduced in mdx52 mice at 50% contrast. The complex ERG alterations and disturbed contrast vision in mdx52 mice include features observed in DMD patients and suggest altered photoreceptor-to-bipolar cell transmission possibly affecting contrast sensitivity. The mdx52 mouse is a relevant model to appraise the roles of retinal dystrophins and for preclinical studies related to DMD.